It is now clear that antiretroviral therapy (ART) alone does not eradicate HIV: Even after more than 15 years of intensive and continuous therapy, the spread of the virus resumes within a few weeks upon cessation of ART in all but exceptional cases. In virally suppressed subjects, only about one in a million resting CD4+ T cells contain latent proviruses capable of producing replication-competent virus. Quantifying cells harboring latent HIV is critical to evaluating strategies to eliminate them, but the low frequency of these cells makes this extremely challenging. With an increasing number of novel and innovative therapeutic strategies that will be tested in humans to reduce the size of the latent reservoir, there is an urgent need to develop a robust, precise and clinical trial scalable assay that measures the frequency of latently cells infected cells carrying inducible HIV. We have recently developed an assay to measure the magnitude of the latent -and inducible- HIV reservoir, which will fulfill these criteria. This novel assay, named TILDA for Tat/rev Induced Limiting Dilution Assay, measures the frequency of cells with multiply spliced HIV RNA upon maximal cellular activation with PMA/ionomycin. Importantly, our assay does not require extraction of viral nucleic acids, which makes TILDA suitable for high throughput studies. TILDA requires only 10mL of blood, is extremely reproducible (coefficient of variation = 0.2), covers a wide dynamic range of reservoir size (over 3 logs) and can be completed in two days. The objective of this proposal is to demonstrate that TILDA can be used to precisely measure the frequency of latently infected cells that persist in virally suppressed subjects. We will use TILDA to measure the size of the reservoir in 36 subjects on suppressive ART and will correlate these values with those measured by the gold standard method (quantitative viral outgrowth assay, Q-VOA) in two laboratories with strong expertise in this assay (Dr. Siliciano and Dr. Markowitz, collaborators). In Specific Aim 1, we will determine if the frequency of reservoir cells measured by TILDA correlates with those measured by Q-VOA. We hypothesize that these frequencies will correlate, but that TILDA will provide a better estimate of the size of the pool of latently infectd cells. In Specific Aim 2, we will use TILDA to assess the relative proportion of cells with integrated HIV DNA that can be induced to produce HIV in subjects who started ART during the acute and the chronic stages of HIV infection. We predict that a large fraction of the integrated genomes will not be inducible in individuals who started ART during chronic infection. In Specific Aim 3, we will use TILDA to measure the size of the latent HIV reservoir in distinct memory CD4 T cell subsets, with the hypothesis that the more differentiated subsets (effector memory cells) will carry a larger proportion of defective viruses. The overarching goal of this project is to demonstrate that TILDA can be used universally in a clinical setting to precisely measure the size of the latent reservoir and assess the efficacy of eradication strategies.